Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells

Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (Delta SUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P < 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.